mouse anti brca2 Search Results


mouse α brca2  (Merck & Co)
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Merck & Co mouse α brca2
(A) A published gene essentiality screen in <t>BRCA2-proficient</t> and -deficient RPE1 cells ( Adam et al. , 2021 ) was mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA2-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) DLD1 WT and BRCA2 -/- cells were infected with empty vector (CTRL) or EXO1 -targeting sgRNA and viability was measured using CellTiter-Glo (n=3, mean+SD, paired t-test). (C) Lysates of the indicated DLD1 cells indicated in analysed by western blotting. Samples were run on the same gel, dashed line indicates removal of non-relevant lanes post-imaging. (D) H1299 cells carrying a doxycycline-inducible BRCA2 shRNA were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay in absence or presence of 10 μg/mL doxycycline (n=3, mean+SD, paired t-test). (E) Western blot analysis of the lysates of the H1299 TetOn sh BRCA2 cells studied in .
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(A) A published gene essentiality screen in BRCA2-proficient and -deficient RPE1 cells ( Adam et al. , 2021 ) was mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA2-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) DLD1 WT and BRCA2 -/- cells were infected with empty vector (CTRL) or EXO1 -targeting sgRNA and viability was measured using CellTiter-Glo (n=3, mean+SD, paired t-test). (C) Lysates of the indicated DLD1 cells indicated in analysed by western blotting. Samples were run on the same gel, dashed line indicates removal of non-relevant lanes post-imaging. (D) H1299 cells carrying a doxycycline-inducible BRCA2 shRNA were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay in absence or presence of 10 μg/mL doxycycline (n=3, mean+SD, paired t-test). (E) Western blot analysis of the lysates of the H1299 TetOn sh BRCA2 cells studied in .

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) A published gene essentiality screen in BRCA2-proficient and -deficient RPE1 cells ( Adam et al. , 2021 ) was mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA2-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) DLD1 WT and BRCA2 -/- cells were infected with empty vector (CTRL) or EXO1 -targeting sgRNA and viability was measured using CellTiter-Glo (n=3, mean+SD, paired t-test). (C) Lysates of the indicated DLD1 cells indicated in analysed by western blotting. Samples were run on the same gel, dashed line indicates removal of non-relevant lanes post-imaging. (D) H1299 cells carrying a doxycycline-inducible BRCA2 shRNA were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay in absence or presence of 10 μg/mL doxycycline (n=3, mean+SD, paired t-test). (E) Western blot analysis of the lysates of the H1299 TetOn sh BRCA2 cells studied in .

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Infection, Plasmid Preparation, Western Blot, Imaging, shRNA, Transduction, Clonogenic Cell Survival Assay

(A) RPE1 hTERT TP53 -/- BRCA1 -/- cells expressing Cas9, either TP53BP1 +/+ or TP53BP1 -/- , were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay (n=3, mean+SD, **p<0.01, paired t-test). Western blot of lysates shown in supplemental figure 2A. (B) Indicated RPE1 cell lines were incubated with EdU and PARGi for 30 minutes, followed by IF microscopy to analyse PAR formation in EdU-positive cells. A representative of two independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (C) As in , but for BRCA1 -mutated MDA-MB-436 cells, either WT or reconstituted with BRCA1 cDNA, infected with empty vector (CTRL) or EXO1 -targeting sgRNA. A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (D) As in , but for DLD1 cells, either WT or BRCA2 -/- . A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney).

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) RPE1 hTERT TP53 -/- BRCA1 -/- cells expressing Cas9, either TP53BP1 +/+ or TP53BP1 -/- , were transduced with an AAVS1-targeting (CTRL) or EXO1 -targeting sgRNA, followed by a clonogenic survival assay (n=3, mean+SD, **p<0.01, paired t-test). Western blot of lysates shown in supplemental figure 2A. (B) Indicated RPE1 cell lines were incubated with EdU and PARGi for 30 minutes, followed by IF microscopy to analyse PAR formation in EdU-positive cells. A representative of two independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (C) As in , but for BRCA1 -mutated MDA-MB-436 cells, either WT or reconstituted with BRCA1 cDNA, infected with empty vector (CTRL) or EXO1 -targeting sgRNA. A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney). (D) As in , but for DLD1 cells, either WT or BRCA2 -/- . A representative of three independent experiments is shown, black line indicates median (****p<0.0001, Mann-Whitney).

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Expressing, Transduction, Clonogenic Cell Survival Assay, Western Blot, Incubation, Microscopy, MANN-WHITNEY, Infection, Plasmid Preparation

(A) Results of a gene essentiality screen in BRCA1-proficient and -deficient cells ( Adam et al. , 2021 ) were mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA1-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) Indicated RPE1 cells were transfected with a control (siCTRL) or EXO1 -targeting siRNA, followed by treatment with IR and subsequent IF microscopy to analyse either RAD51 or RAD52 foci formation. Left panel shows quantification (n=3, mean+SD, *p<0.05, paired t-test) and right panel shows representative microscopy images of eGFP-RAD52 foci. Western blot of lysates shown in supplemental figure 3A. (C) Clonogenic survival assay of Cas9-expressing RPE1 hTERT TP53 -/- BRCA1 -/- cells that were transduced to express the indicated sgRNAs (n=3, mean+SD, *p<0.05, **p<0.01, one-way ANOVA, post-hoc Dunnett’s, compared to CTRL). Western blot of lysates shown in supplemental figure 3D. (D) HEK 293T cells carrying the DSB-Spectrum_V3 reporter, either WT or EXO1 -/- , were transfected with indicated siRNAs, followed by a second round of transfection with a Cas9 cDNA and BFP sgRNA targeting the reporter locus. Next, cells were analysed by flow cytometry to quantify repair by the indicated pathways (n=4, mean±SEM, *p<0.05, ratio paired t-test). Western blot of lysates shown in supplemental figure 3E. (E) Nuclear γH2AX intensity in S-phase (EdU + ) cells analysed by IF microscopy in BRCA2 -/- cells infected with empty vector or sgEXO1 . A representative of two independent experiments is shown, black line indicates median.

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) Results of a gene essentiality screen in BRCA1-proficient and -deficient cells ( Adam et al. , 2021 ) were mined to extract the CCA scores for the indicated genes. A higher CCA score indicates a unique essentiality in BRCA1-deficient cells compared to proficient cells. Dashed line indicates the cut-off for a significant CCA score (based on Adam et al. , 2021 ). (B) Indicated RPE1 cells were transfected with a control (siCTRL) or EXO1 -targeting siRNA, followed by treatment with IR and subsequent IF microscopy to analyse either RAD51 or RAD52 foci formation. Left panel shows quantification (n=3, mean+SD, *p<0.05, paired t-test) and right panel shows representative microscopy images of eGFP-RAD52 foci. Western blot of lysates shown in supplemental figure 3A. (C) Clonogenic survival assay of Cas9-expressing RPE1 hTERT TP53 -/- BRCA1 -/- cells that were transduced to express the indicated sgRNAs (n=3, mean+SD, *p<0.05, **p<0.01, one-way ANOVA, post-hoc Dunnett’s, compared to CTRL). Western blot of lysates shown in supplemental figure 3D. (D) HEK 293T cells carrying the DSB-Spectrum_V3 reporter, either WT or EXO1 -/- , were transfected with indicated siRNAs, followed by a second round of transfection with a Cas9 cDNA and BFP sgRNA targeting the reporter locus. Next, cells were analysed by flow cytometry to quantify repair by the indicated pathways (n=4, mean±SEM, *p<0.05, ratio paired t-test). Western blot of lysates shown in supplemental figure 3E. (E) Nuclear γH2AX intensity in S-phase (EdU + ) cells analysed by IF microscopy in BRCA2 -/- cells infected with empty vector or sgEXO1 . A representative of two independent experiments is shown, black line indicates median.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Transfection, Microscopy, Western Blot, Clonogenic Cell Survival Assay, Expressing, Flow Cytometry, Infection, Plasmid Preparation

(A) Model of the mechanism causing synthetic lethality between BRCA1-deficiency and EXO1 loss. (B) Whole genome sequencing data of pan cancer tumour samples ( Martínez-Jiménez et al. , 2022 ) was analysed to quantify the number of genetic scars indicative of DSB-repair by SSA, here defined as deletions flanked by homologous sequences of >10bp. CHORD analysis was used to classify samples as HR-proficient or HR-deficient, either BRCA1-type or BRCA2-type ( Nguyen et al. , 2020 ) (****p<0.0001, kolmogornov-smirnov). (C) Tumour samples from a pan-cancer cohort were binned based on SSA scar count, and the EXO1 expression was plotted for each tumour sample. (D) EXO1 expression levels in BRCA1 WT or BRCA1 mutant pan-cancer tumour samples.

Journal: bioRxiv

Article Title: EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

doi: 10.1101/2023.02.24.529205

Figure Lengend Snippet: (A) Model of the mechanism causing synthetic lethality between BRCA1-deficiency and EXO1 loss. (B) Whole genome sequencing data of pan cancer tumour samples ( Martínez-Jiménez et al. , 2022 ) was analysed to quantify the number of genetic scars indicative of DSB-repair by SSA, here defined as deletions flanked by homologous sequences of >10bp. CHORD analysis was used to classify samples as HR-proficient or HR-deficient, either BRCA1-type or BRCA2-type ( Nguyen et al. , 2020 ) (****p<0.0001, kolmogornov-smirnov). (C) Tumour samples from a pan-cancer cohort were binned based on SSA scar count, and the EXO1 expression was plotted for each tumour sample. (D) EXO1 expression levels in BRCA1 WT or BRCA1 mutant pan-cancer tumour samples.

Article Snippet: The following primary antibodies were used for western blotting: Mouse α 53BP1 (BD 612522; 1:1,500), Rabbit α BLM (Abcam ab2179; 1:2,000), Mouse α BRCA1 (Merck OP92; 1:1,000), Rabbit α BRCA1 (Millipore 07-434; 1:2,000), Mouse α BRCA2 (Merck OP95; 1:1,000), Mouse α CTIP (Millipore MABE1060; 1:1,000), Rabbit α EXO1 (Abcam ab95068; 1:1,000), Rabbit α EXO1 (Millipore ABE1354; 1:1,000), Mouse α GFP (Sigma #11814460001; 1:1000), Rabbit α MRE11 (1:3,000; de Jager et al., 2001), Mouse α TUBULIN (Sigma T6199; 1:5,000), and Mouse α RAD52 (Santa-Cruz sc-365341; 1:100).

Techniques: Sequencing, Expressing, Mutagenesis